The ability to generate hematopoietic stem cells (HSCs) from human pluripotent stem cells (PSCs), embryonic (hESCs) and induced pluripotent stem cells (hiPSC) would enable the production of unlimited numbers of patient-matched stem cells for transplantation and the derivation of novel in vitro models for studying human hematopoietic development and disease. Numerous studies have shown that it is possible to derive hematopoietic lineage cells from hPSCs either by co-culturing them with stromal cells in serum-based media, or by directing their differentiation with specific morphogens in defined serum-free media (Chadwick et al., 2003; Davis et al., 2008; Kaufman et al., 2001; Kennedy et al., 2007; Ledran et al., 2008; Ng et al., 2005; Pick et al., 2007; Vodyanik et al., 2006; Yu et al., 2010; Zambidis et al., 2005). While these approaches yield a broad spectrum of hematopoietic progenitors, transplantation of the progeny from such cultures into immunocompromised mice has typically resulted in low levels of engraftment often restricted to the myeloid lineages (Lu et al., 2009; Tian et al., 2006; Wang et al., 2005). These findings suggest that the conditions used for hematopoietic differentiation do not support the development of HSCs. A major factor contributing to the failure in generating HSCs from hPSCs is the complexity of the embryonic hematopoietic system, which consists of at least two distinct programs, only one of which gives rise to HSCs.
HSCs are generated from the definitive hematopoietic program and develop from a specialized population of endothelial cells, known as hemogenic endothelium (HE; (Dzierzak and Speck, 2008)). In the mouse, HE is specified at different sites within the developing vasculature, of which the best characterized is the para-aortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) region found in the caudal portion of the embryo. The mouse HE is characterized by expression of a panel of hematopoietic and endothelial markers, including VE-cadherin (VE-cad), Sca-1, c-Kit, CD34, Runx1, Scl, Gata2 and Lmo2 (reviewed in (Dzierzak and Speck, 2008)). HSCs are first detectable in the AGM region at E10.5 and are characterized by the acquisition of low CD45 expression in addition to the above set of markers (Bertrand et al., 2005; Taoudi and Medvinsky, 2007; Yokomizo and Dzierzak, 2010). The human P-Sp/AGM region is also a site of definitive hematopoiesis as it contains progenitors that express markers indicative of HE and hematopoietic development including CD31, CD34, CD45, C-KIT, SCL, C-MYB, GATA2 and GATA3 (Labastie et al., 1998; Marshall et al., 1999; Oberlin et al., 2002; Tavian et al., 2001) and by gestational day 32 have in vivo multilineage repopulating capacity (Ivanovs et al., 2012).
Definitive hematopoiesis is preceded by an earlier, yolk sac (YS) restricted program, known as primitive hematopoiesis, that is characterized by the production of primitive erythrocytes, macrophages and megakaryocytes (reviewed in (Palis et al., 2010). Most evidence indicates that primitive hematopoiesis is restricted in potential and does not have the capacity to generate HSCs or lymphoid cells, but recent studies have shown that the YS can generate lymphoid progenitors prior to or in the absence of circulation (Rhodes et al., 2008; Yoshimoto et al., 2011; Yoshimoto et al., 2012). Further characterization of these yolk sac populations however, revealed that the lymphoid cells developed from a VE-cad+CD41− HE-like progenitor, distinct from the VE-cad− CD41+ primitive hematopoietic progenitors (Yoshimoto et al., 2011; Yoshimoto et al., 2012). These findings indicate that the YS displays both primitive and definitive hematopoietic potential and that the two populations develop from distinct progenitors.
Most studies to-date support the interpretation that lineage development from PSCs recapitulates lineage commitment in the embryo (Murry and Keller, 2008). Thus, the generation of HSCs from PSCs will depend on establishing culture conditions that not only promote HE development, but also on methods to identify these progenitors as they are specified. In an earlier study, we used T cell potential to map the onset of definitive hematopoiesis in mouse ESC differentiation cultures, and demonstrated that this program initiates from a Flk-1+ Sox17+ progenitor that emerged 48 hours following the onset of primitive hematopoiesis (Irion et al., 2010). Several studies have demonstrated that it is possible to generate T lymphocytes from hESCs (Galic et al., 2006; Timmermans et al., 2009).